Virulence of wheat leaf rust (Puccinia triticina Eriks.) and Lr resistance genes in wheat cultivars in the Slovak Republic in the years 2016–2019

In the years 2016 to 2019 the virulence of the wheat leaf rust population in Slovakia was tested on Thatcher near-isogenic lines Lr1, Lr2a, Lr2b, Lr2c, Lr3a, Lr9, Lr10, Lr11, Lr13, Lr15, Lr17a, Lr19, Lr21, Lr23, Lr24, Lr26 and Lr28. A total of 111 rust isolates were analysed. Resistance genes Lr19 and Lr9 were effective to most of the tested isolates. Average virulence frequency to these resistance genes was only 1%. The gene Lr24 conditioned resistance to 92% of the tested rust isolates, gene Lr28 to 86% of the tested rust isolates. Low virulence frequency was also recorded to Lr2a (13%), Lr2b and Lr2c (both 12%). Average virulence to other tested Lr genes exceeded 82%. Thirty-eight winter wheat cultivars registered in the Slovak Republic were tested for the presence of Lr10, Lr19, Lr24, Lr26, Lr28, Lr34 and Lr37 by molecular markers. The gene Lr34 was the most frequent one in the tested cultivars, followed by Lr37 (both genes for adult plant resistance). The most widespread pathotypes in separate years and leaf rust severity in thirty-eight cultivars are also recorded.


Introduction
Wheat leaf rust caused by Puccinia triticina Eriks is considered a serious disease worldwide. This pathogen may cause considerable yield losses particularly in southern and eastern parts of the Slovak Republic during warm and dry years. Resistance breeding may be the most economic means to control the potential damages caused by such pathogen. The knowledge of the virulence in the domestic leaf rust population as well as knowledge and availability of effective sources of resistance are preconditions for successful resistance breeding. This paper contains virulence data in the leaf rust population and data on leaf rust resistance genes (Lr) in selected wheat cultivars together with some data on the same topic published earlier. The data should contribute to the above-mentioned aim (Hanzalová et al. , 2005(Hanzalová et al. , , 2012(Hanzalová et al. , , 2016.

Materials and methods
Collections of wheat leaf rust from different cultivars were obtained from the variety trials located across the country and organized by The Central Control and Testing Institute in Agriculture in Slovakia. The initial source of inoculum was a dry leaf containing one or more pustules. The inoculation was performed by rubbing the leaves between fingers moistened with water suspension of urediniospores. Inoculated plants were sprayed with water and incubated in closed glass cylinders for two days. The second leaf of seedlings of the susceptible wheat cultivar Michigan Amber was inoculated. Plants were then kept in open cylinders in the greenhouse at a temperature of 18-22 °C with supplemental (16 h/d) illumination with fluorescent tubes. When chlorotic spots appeared on inoculated leaves, a leaf segment with one developing uredinium of each rust sample was transferred to a Petri dish with 15 ml of water and kept in the greenhouse until urediniospores developed. Single uredinial isolates were increased on seedlings of the cv. Michigan Amber Communicated by M. Taylor.
1 3 using the same methods of inoculation as described above. Near-isogenic lines with Lr genes were inoculated with the spore multiplication by the same methods as described above. The inoculation of seedlings was carried out with urediniospores (approx 30 μl/50 seedlings in one pot). Infection types were evaluated according to Stakman et al. (1962). Avirulence was characterized by infection types 0,;, 1, 1-2, 2, virulence by infection of type 3. The frequency of rust isolates virulent to the differentials was expressed in percentages. Thatcher near-isogenic lines (NILs) with single Lr genes approved as leaf rust differentials by participants in the international COST 817 Action (Mesterházy et al. 2000) were used as differentials. In addition Lr10 and Lr13 were also included. The seeds of the NILs were supplied by Dr J. Kolmer to the Cereal Research Non-Profit Company in Szeged, Hungary, where it was subsequently multiplied and distributed to participants of the COST 817 action.
In the years 2016-2019 thirty-eight wheat cultivars registered in Slovakia were tested in the National Agricultural and Food Centre in Piešťany (SK) in a naturally infected field. Scoring was carried out three times according to the progress of the infection, symptoms disease severity were evaluated by field scale 1-9 (1 low, 9 high disease severity).
Molecular DNA markers were used to analyse the studied wheat cultivars registered in the Slovak Republic for the presence of selected leaf rust resistance genes (Table 1). DNA was extracted from the second wheat leaf using a DNeasy Plant Mini kit (Qiagen, Germany). The quantity and purity of DNA was measured spectrophotometrically with a NanoDrop-1000 Spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA, USA). The presence of genes Lr10, Lr19, Lr24, Lr26, Lr28, Lr34 and Lr37 was determined by the use of PCR assays with published primers for markers linked to these genes (Gultyaeva et al. 2009;Prins et al. 2001;Schachermayr et al.1995;De Froidmont et al. 1998;Cherukuri et al. 2005;Lagudah et al. 2006;Helguera et al. 2003). PCR amplifications using specific primers were carried out in PTC-200 thermal cycler (MJ-Research). The primers, amplification conditions and size of PCR products indicating the probable presence of analysed resistance genes are listed in Table 1. The amplificated products were separated by electrophoresis in 2% agarose gels, stained with ethidium bromide and visualized under UV light. 50 bp and 100 bp DNA Ladder (Invitrogen) were used as molecular weight markers. Thatcher near-isogenic lines with the corresponding Lr genes were included as positive controls and sterile water and cultivar Thatcher without Lr genes as negative control.

Virulence in the leaf rust population and pathotypes
In the years 2016-2019 very low average virulence to Lr9 and Lr19 (1%) and Lr24 (8%) was recorded followed by Lr2a, Lr2b and Lr2c (13% and 12%) and Lr28 (14%) ( Table 2). Average virulence frequency to other tested Lr genes exceeded 82%. The fluctuation of the virulence frequency in single years was relatively low except for Lr2a, Lr2b and Lr2c in the year of 2017 with virulence frequency 33%. No distinct trend was observed in the fluctuation of the virulence frequency to other Lr genes.
In the years 2016, 2017, 2018 and 2019 the number of determined pathotypes on 17 differential near-isogenic lines was 10, 16, 5 and 9, respectively. In the same years the most frequent pathotypes were virulent to 10 differentials except for 2016 (9 differentials) ( Table 3). Two rust isolates in the year 2017 and the same number in 2019 were virulent to 12

Analyses of the presence of Lr genes by molecular markers
Out of thirty-eight winter wheat cultivars, fourteen cultivars possessed the gene Lr34, twelve of the cultivars possessed the gene Lr37, single cultivars possessed the genes Lr10, Lr19 and Lr26. Markers for the genes Lr24 and Lr28 were not found in any of the tested cultivars (Table 4). The segregation of genes Lr34 and Lr37 was observed in cultivar Juno. The combination of markers for two genes was found in four cultivars, in cultivar PS Puqa markers for genes Lr10 and Lr37, in cultivar IS Signum marker genes Lr34 and Lr37, in cultivar PS Agneska genes Lr26 and Lr37 and in cultivar Juno genes Lr34 and Lr37 in heterozygous stage.
In the previous study (Hanzalová et al. 2020) of nineteen recently registered Czech cultivars the gene Lr37 prevailed, followed by genes Lr10, Lr24, Lr28, Lr1 and Lr26. The genes Lr19 and Lr34 were not determined. Serfling et al. (2011) recorded similar ranking of the above-mentioned Lr genes in the analysis of 115 German cultivars by molecular markers. The authors of the above-mentioned publication found the gene Lr37 in 48 cultivars, gene Lr10 in 35 cultivars, gene Lr26 in 14 cultivars and gene Lr34 only in 2 cultivars. Also within 37 winter wheat cultivars registered in Slovakia Hanzalová et al. (2016) found the most frequent gene Lr37 (16 cultivars), followed by the genes Lr34 (6 cultivars), Lr10 (5 cultivars) and Lr26 (4 cultivars). On the contrary in the study of 38 Slovak cultivars that is described in this paper, the gene Lr34 prevailed (14 cultivars), followed by Lr37 (12 cultivars) and genes Lr10, Lr19 and Lr26 only with single cultivar for each. The gene Lr34 prevailed (in 7 genotypes) also within the group of 46 Chinese landraces (Zhang et al. 2019). Genes Lr37 and Lr34 condition adult plant resistance and are partially effective. Within 136 African wheat genotypes, Kankwatsa et al. (2019) found 46 genotypes with one or more adult plant resistance genes Lr34, Lr46, Lr67, Lr68 and Lr74, which can confer high levels of  (Hiebert et al. 2016). The gene Lr10 can be useful in combination with other resistance genes. The gene Lr26 is predominantly ineffective. In addition to major genes, the minor genes also play an important role in the resistance of wheat in the field.

Resistance of registered cultivars in field trials
Although the scoring of rust severity comes from only one location and not all cultivars were tested for 5 years, recorded data suggest several conclusions valid at least for the given location. Cultivar Bona Vita possessing Lr19 was the most resistant one, in accordance with results of virulence survey. No virulence was recorded in the years 2017, 2018, 2019 and very low (1%) in 2016. Relatively high resistance was recorded in most cultivars possessing the gene Lr34 for adult plant resistance. Of them cv. IS Escoria had even the same high scoring as cv. Bona Vita. That can be hardly attributed exclusively to the gene Lr34.

Cultivar
Average field scoring Availability of data and material The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Seeds are not publicly available until released as commercial genotypes.
Code availability Not applicable.

Conflict of interest
The authors declare that they have no conflicts of interest.
Ethics approval Not applicable.

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